DESCRIPTION: The goal of the proposed research is to develop critically need new drugs against M. tuberculosis. The basic hypothesis is that new drugs against M. tuberculosis can be developed if inhibitors of the enzymes catalyzing transformations involved in cell wall biosynthesis are discovered. In future research, such inhibitors would be modified as necessary so they can reach the target enzymes in TB patients. For the proposed study, eight key enzymatic transformations involved in the cell wall D-arabinofuranose, D-galactofuranose, and L-rhamnose biosynthesis are to be targeted. These eight transformations are not found in humans and hence drugs based on inhibiting them will hopefully not be toxic. To develop these eight drug targets, the genes encoding for their synthesis will be cloned, usually utilizing partial amino acid sequence obtained after enzyme purification. These cloned gene will be used, in a series of molecularly based experiments, to confirm the expectation that their encoded enzyme products are essential for mycobacterial viability. The genes of all enzymes confirmed to be essential for viability will be over-expressed. Such over-expression will allow inhibitor identification to proceed in two complementary fashions. The first involves large scale screening of compound libraries for inhibitors; collaborations are in place at GlaxoWellcome toward this end. In the second approach the mechanism of action of the enzymes including the identification of active sites and essential amino acids will be elucidated. For selected enzymes these studies will be carried to the level of protein crystallization and X-ray analysis with collaborators at the University of St. Andrews. This second approach will allow "rationally" designed inhibitors to be developed.